Francisella tularensis (Ft), Yersinia pestis (Yp), and Bacillus anthracis (Ba) are considered Category A bioweapons due to ease of transmission, low infectious dose and high mortality associated with pneumonic forms of disease, and the fact that all have been intensively studied and developed in bioweapons programs in several countries. There are currently no vaccines against tularemia or plague approved for general human use, rendering mankind at significant risk from the illicit use of Ft and Yp. Our prior studies have shown that a Ft subsp. novicida (Fn) FPI mutant (Fn-iglD) can protect against a pulmonary challenge of Ft subsp. tularensis (Ftt), in both rat and non-human primate (NHP) models of tularemia. This is an extremely promising result, because Fn is naturally avirulent toward humans, and thus a safer basis for a human vaccine. Antibodies against the Yp F1 capsular and LcrV virulence antigens fused into a single polypeptide (F1V) have been shown to be protective against Yp pulmonary challenge in several animal models, including NHP. Moreover, antibodies against the Ba protective antigen (PA) protect against Ba pulmonary challenge. The studies outlined here are designed to optimize the Fn-iglD vaccine platform to provide protection against Yp and Ba by expression of F1V and PA. The Fn-iglD vaccine strain will be engineered to present F1V on its surface and to secrete PA. The efficacy of this vaccine to protect against pulmonary challenge with Ftt and/or Yp, as well as challenge with anthrax lethal toxin (LT) will be measured in the rat model. The result of these studies will be a vaccine with broad efficacy against multiple biothreat agents. The collaborative team at UTSA and UTHSCSA has extensive experience in tularemia, plague, and anthrax vaccine development that will propel the further development of this biothreat vaccine platform. The R21 mechanism is intended to fund ?early and conceptual stages of project development?; upon completion of the experiments outlined in this proposal, this project will be poised for the submission of a well-developed RO1 proposal directed at an effective multivalent biodefense vaccine.